MYCOBACTERIUM TUBERCULOSIS DeltaESX-3 MUTANTS

ABSTRACT

Isolated mutant  Mycobacterium tuberculosis  bacteria comprising a deletion in the ESAT-6 gene cluster region 3 (esx-3 region) are provided, as well as compositions comprising such, methods of production thereof and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 61/645,391, filed May 10, 2012, the contents of which are hereby incorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant number AI026170 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to by number in parentheses. Full citations for the references may be found at the end of the specification. The disclosures of each of these publications, and also the disclosures of all patents, patent application publications and books recited herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

The ESAT-6 gene cluster region 3 (“esx-3 region”, namely, Rv0282 through Rv0292 in Mycobacterium tuberculosis H37Rv) encodes one of five paralogous Esx (type VII) secretion systems within the Mycobacterium tuberculosis genome, and appears to be present in all mycobacterial species sequenced to date (6). Although the specific substrates of this transport system are unknown, extensive previous work, including both saturating transposon mutagenesis studies and attempts to generate deletion mutants through homologous recombination (4-6), has suggested that the esx-3 region of M. tuberculosis is essential for growth in vitro and cannot be deleted. In contrast, it is not required for the growth of the saprophytic mycobacterium M. smegmatis (e.g. see PCT International Application Publication No. WO 2009/008912, Jacobs et al., published Jan. 15, 2009, hereby incorporated by reference in its entirety). In view of the attenuated virulence and TH1 cytokine profile of M. smegmatis Δesx-3 mutants, it would be desirable if a way could be achieved to generate M. tuberculosis Δesx-3 mutants.

The present invention address the need for attenuated M. tuberculosis mutants and related vaccines based on M. tuberculosis Δesx-3 mutants.

SUMMARY OF THE INVENTION

A non-naturally occurring mutant Mycobacterium tuberculosis bacterium, wherein the mutant M. tuberculosis comprises a deletion in the ESAT-6 gene cluster region 3 (esx-3 region) of a genome of the M. tuberculosis bacterium.

The invention also provides a method of producing a mutant Mycobacterium tuberculosis bacterium, wherein the mutant M. tuberculosis comprises a deletion in the esx-3 region of a genome of the M. tuberculosis bacterium, the method comprising deleting a nucleic acid sequence in the esx-3 region of a genome of a M. tuberculosis bacterium.

The invention also provides a composition comprising a mutant Mycobacterium tuberculosis bacterium, wherein the mutant M. tuberculosis comprises a deletion in the esx-3 region of a genome of the M. tuberculosis bacterium and a carrier

The invention also provides a method of eliciting an immune response in a subject comprising administering to the subject a composition comprising a non-naturally occurring mutant Mycobacterium tuberculosis bacterium, or a composition comprising such, wherein the mutant M. tuberculosis comprises a deletion in the esx-3 region of a genome thereof, in an amount effective to elicit an immune response.

The invention also provides a non-naturally occurring mutant Mycobacterium tuberculosis bacterium, wherein the mutant M. tuberculosis comprises a deletion of (i) EsxG, (ii) EsxH, (iii) Esxg and EsxH, (iv) PE5-PPE4, or (v) the four genes PE5 to esxH, of the genome of the M. tuberculosis bacterium. The invention also provides a composition comprising such.

The invention also provides a method of eliciting an immune response in a subject comprising administering to the subject a composition comprising a non-naturally occurring mutant Mycobacterium tuberculosis bacterium, or the composition comprising such, wherein the non-naturally occurring mutant M. tuberculosis comprises a deletion of (i) esxG, (ii) esxH, (iii) esxg and esxH, (iv) PE5-PPE4, or (v) the four genes PE5 to esxH, of a genome of the M. tuberculosis bacterium, in an amount effective to elicit an immune response.

Additional objects of the invention will be apparent from the description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. H37Rv Δesx-3 transductants growing on 7H10 supplemented with hemin 100 μM. M. tuberculosis H37Rv was incubated at 37° C. with a specialized transducing phage harboring a construct designed to delete the entire M. tuberculosis esx-3 region (Rv0282-Rv0292) and replace the locus with a sacB-hygromycin resistance cassette, as described in the text. Transductions were plated onto 7H10 agar supplemented with hygromycin at 50 micrograms per ml, and hemin at 100 μM. Three colonies were obtained, initially observed at ˜4 weeks of incubation at 37° C., and here photographed at ˜7 weeks of incubation.

FIGS. 2A-2D. A-B: To confirm that the colonies obtained were esx-3 deletion mutants, genomic DNA was prepared from H37Rv Δesx-3 transductants (#1-#3) and H37Rv wild-type. The DNA was digested with BamHI and, after separation by agarose gel electrophoresis, Southern blots were performed, using as probes sequences immediately flanking the deleted esx-3 region: a 900-bp left flank probe amplified with primers esx-3LL and esx-3LR (A) or a 867-bp right flank probe amplified with primers esx-3RL and esx-3RR (B). In (C) and (D) size differences are the result of elimination of BamHI sites from the chromosome by the extensive deletion, while the inserted sacB-hygromycin resistance cassette contains no BamHI sites (shown schematically).

FIG. 3. PCR analysis of H37Rv esx-3 deletion clones. A-B. Genomic DNA prepared from H37Rv Δesx-3 clones, or H37Rv wt, was used as template in PCR reactions to amplify products using three different sets of primers. Primer set P1+P2+P3 includes a common left hand primer, P1 (to the left of flank sequences cloned to make the deletion construct, binding in both wt and mutant), and complementary primers P2 (binds within the esx-3 region, in wild-type only) and P3 (binds to plasmid sequences, in mutant only). Primer set P4+P5+P6 includes a common right hand primer, P4 (to the right of cloned flank sequences, binding in both wt and mutant), and complementary primers P5 (binds within the esx-3 region, in wild-type only) and P6 (binds to plasmid sequences in mutant only). Finally, primers P7+P8 are within the deleted esx-3 region, and so amplify a product in wild-type only. Schematics of H37Rv wt (A) and H37Rv Δesx-3 (B) chromosomal loci indicate the locations of the three primer sets, and the expected product sizes for wild-type and mutant. C. PCR products using mutant or wild-type genomic DNA as template, or water as negative control, were subjected to agarose gel electrophoresis. Lane designations: 1, H37Rv Δesx-3 clone #1; 2, H37Rv Δesx-3 clone #2; 3, H37Rv Δesx-3 clone #3; wt, H37Rv wt; H₂O, water as negative control.

FIG. 4. Hemin enhances the growth of H37Rv Δesx-3 on solid medium. Cultures of H37Rv wt growing in 7H9 liquid medium and H37Rv Δesx-3 growing in 7H9 supplemented with hygromycin 50 μg per ml and hemin (100 μM) were pelleted by centrifugation and washed twice with 7H9+0.05% Tween 80 (without supplements), then serially diluted in 7H9+Tween, and 10 μl spots were placed onto 7H10, or 7H10 plus hemin 100 μM and allowed to dry. Plates were photographed after 14 days of incubation at 37° C.

FIG. 5. Hemin enhances growth of H37Rv Δesx-3 in liquid medium. Cultures of H37Rv wt (black symbols) and H37Rv Δesx-3 (gray symbols), both growing in 7H9 medium supplemented with hemin 100 μM (also with hygromycin 50 μg per ml for the esx-3 mutant) were pelleted by centrifugation, washed three times with PBS+tyloxapol 0.05% and resuspended in PBS-tyloxapol, after which each suspension was adjusted to OD₆₀₀=1. Washed bacteria (250 μl) were added to 10 ml of 7H9 complete medium containing tyloxapol 0.05%, either unsupplemented (squares) or supplemented with hemin at 1 (triangles), 10 (inverted triangles) or 100 (diamonds) micromolar. Cultures were incubated with shaking at 37° C., and aliquots were removed at the indicated time points to measure the OD₆₀₀.

FIG. 6. H37Rv Δesx-3 is significantly attenuated in SCID mice. SCID mice (7-8 mice per group) were injected intravenously via the tail vein with 10⁷ cfu of H37Rv wt (blue squares) or H37RvΔesx-3 (red inverted triangles) or with 10⁴ cfu of H37Rv wt (blue circles) or H37RvΔesx-3 (red diamonds). (cfu estimated by measurement of OD₆₀₀). Prior to injection, bacteria (both wt and mutant) were cultured in 7H9 medium containing tyloxapol 0.05% and hemin 100 μM; medium for the mutant also contained hygromycin 50 μg per ml. Bacteria were washed three times with PBS-0.05% Tween 80 prior to injection. Survival was monitored over time. Actual doses per plating of inocula are indicated.

FIG. 7. H37Rv Δesx-3 is also significantly attenuated in RAG(−/−) and MyD88(−/−) mice. RAG(−/−) mice (panel A.) or MyD88(−/−) mice (panel B) were infected intravenously with 10⁷ cfu of H37Rv wt (blue squares) or H37Rv Δesx-3 (red inverted triangles), as described for the infection of SCID mice (FIG. 6). Again, cfu was estimated by measurement of OD₆₀₀. Survival was monitored over time.

FIG. 8: Mid- to late-log phase cultures of the indicated bacterial strains were washed in PBS+0.05% tyloxapol, resuspended in volumes to give equivalent OD₆₀₀ values, then serially diluted in PBS-tyoxapol to give approximately 300 colonies per 100 μl, and 100 μl aliquots were plated onto each plate. The strains included H37Rv Δesx-3, H37Rv Δesx-3 attB::pYUB1335 (complemented with a cosmid containing the esx-3 region of M. tuberculosis H37Rv), H37Rv Δesx-3 attB::pYUB2076 (complemented with a cosmid containing the paralogous esx-3 region of M. smegmatis), H37RvΔesxG (Rv0287). H37RvΔesxH (Rv0288), and H37RvΔmbtB (Rv2383c). It can be observed that growth of the Δesx-3, ΔesxG and ΔesxH mutants is not supported in 7H10 media, but significant growth is observed on 7H10 media supplemented with mycobactin J (Allied Monitor) at 200 ng/ml. In contrast, the ΔmbtB mutant, predicted from the literature to be deficient in mycobactin synthesis, is able to grow with mycobactin J supplement in amounts as low as 2 ng/ml. The ΔesxG mutant is unique among these mutants in the ability to grow on 7H10 supplemented with additional iron and zinc: in this case ferric ammonium citrate (FAC) at 250 μg/ml and zinc sulfate (ZnSO₄) at 10 μg/ml.

FIG. 9: PCR Screen for H37Rv Δesx-3 complemented with a cosmid encoding the M. smegmatis esx-3 region (pYUB2076).

DETAILED DESCRIPTION OF THE INVENTION

A non-naturally occurring mutant Mycobacterium tuberculosis bacterium, wherein the mutant M. tuberculosis comprises a deletion in the ESAT-6 gene cluster region 3 (esx-3 region) of a genome of the M. tuberculosis bacterium. The non-naturally occurring mutant Mycobacterium tuberculosis bacterium is a mutant by virtue of the deletion in the ESAT-6 gene cluster region 3.

In an embodiment, the M. tuberculosis in which the deletion in the esx-3 region is effected is one of the following: Mycobacterium tuberculosis H37Rv, BTB05-552, BTB05-559, CDC1551, CTR1-2, F11, H37, H37Ra, HN878, KZN 1435, KZN 4207, KZN R506, KZN V2475, R1207, RGTB327, S96-129, X122, ‘98-R604 INH-RIF-EM’, 02_(—)1987, 210, 94_M4241A, C, CDC1551A, CPHL_A, CTR1-4, EAS054, GM 1503, K85, KZN 605, OSDD071, OSDD504, OSDD518, SUMu001, SUMu002, SUMu003, SUMu004, SUMu005, SUMu006, SUMu007, SUMu008, SUMu009, SUMu010, SUMu011, SUMu012, T17, T46, T85, T92, W-148, str. Haarlem, 210_(—)16C10, 210_(—)16C2_(—)24C1, 210_(—)16C2_(—)24C2, 210_(—)32C4, 210_(—)4C15, 210_(—)4C15_(—)16C1, 210_(—)4C15_(—)16C1_(—)48C1, 210_(—)4C15_(—)16C1_(—)48C2, 210_(—)4C15_(—)16C1_(—)56C1, 210_(—)4C15_(—)16C1_(—)56C2, 210_(—)4C31, 210_(—)4C31_(—)16C1, 210_(—)4C31_(—)16C1_(—)24C1, 210_(—)4C31_(—)16C1_(—)40C1, 210_(—)4C31_(—)16C2, 210_(—)8C1, 210_(—)8C6, BC, CTR1-3, H37Rv_(—)2009, NJT210GTG, str. Erdman=ATCC 35801, str. Erdman WHO, CCDC5079, CCDC5180, RGTB423, UT205, CTR1-1, H37RvAE, H37RvCO, H37RvHA, H37RvJO, H37RvLP, H37RvMA, LAM7, NCGM2209, RGTB306, WX1, WX3, XDR1219, XDR1221, str. Beijing/W BT1, or str. Erdman (ATCC 35801). In a preferred embodiment, the M. tuberculosis bacterium is an H37Rv strain. Also provided is a mycobacterium in which the esx-3 region is deleted, wherein the mycobacterium is a M. bovis or M. bovis BCG.

In embodiments the M. tuberculosis in which the esx-3 region deletion is effected is an MDR-TB or an XDR-TB. In embodiments the M. tuberculosis in which the esx-3 region deletion is resistant to, or is suspected of being resistant to kanamycin, isoniazid and/or rifampicin, an aminoglycosides (e.g., amikacin), a polypeptide (e.g., capreomycin, viomycin, enviomycin), a fluoroquinolone, (e.g., ciprofloxacin, levofloxacin, moxifloxacin), and/or a thioamide (e.g. ethionamide).

In an embodiment, the genome in which the deletion is effected has the same sequence as a genome set forth in NCBI Reference Sequence NC_(—)002755.2, NC_(—)009565.1, NC_(—)009525.1, NC_(—)000962.2, NC_(—)012943.1, NZ_ACVS00000000.2, NZ_CM000787.2, CP001662.1, NC_(—)016768.1, NZ_ACVU00000000.2, NZ_CM000789.2, NZ_ACVT00000000.2, NZ_CM000788.2, NC_(—)017026.1, NZ_ABVM00000000.1, NZ_ABLM00000000.1, NZ_ADAB00000000.1, NZ_ABLL00000000.1, NZ_AAKR00000000.1, NZ_AAKR00000000.1, AELF00000000.1, AELF00000000.1, NZ_ABOV0000000.1, NZ_ABQG00000000.1, NZ_AAYK00000000.1, NZ_ACHQ00000000.1, NZ_ABGN00000000.2, NZ_ADHQ00000000.1, NZ_ADHR00000000.1, NZ_ADHS00000000.1, NZ_ADHT00000000.1, NZ_ADHU00000000.1, NZ_ADHV00000000.1, NZ_ADHWO0000000.1, NZ_ADHX00000000.1, NZ_ADHY00000000.1, NZ_ADHZ00000000.1, NZ_ADIA00000000.1, NZ_ADIB00000000.1, NZ_ABQH00000000.1, NZ_ACHO00000000.1, NZ_ABOWO0000000.1, NZ_ABLN00000000.1, or NZ_AASN00000000.1.

In an embodiment, the deletion comprises less than the complete esx-3 region. In an embodiment, the deletion comprises one or more of genes Rv0282, Rv0283, Rv0284, Rv0285, Rv0286, Rv0287, Rv0288, and Rv0292. In a preferred embodiment, the deletion in the esx-3 region renders the resultant recombinant M. tuberculosis less virulent than the wildtype. In an embodiment, the deletion comprises all of genes Rv0282, Rv0283, Rv0284, Rv0285, Rv0286, Rv0287, Rv0288. Rv0289, Rv0290, Rv0291 and Rv0292. In an embodiment, the deletion comprises genes corresponding to Rv0282, Rv0283, Rv0284, Rv0285, Rv0286, Rv0287, Rv0288, Rv0289, Rv0290, Rv0291 and Rv0292. In a preferred embodiment, the mutant M. tuberculosis comprises a deletion of the esx-3 region (Rv0282 through Rv0292) of the genome. In an embodiment, the deletion comprises all of genes Rv0282, Rv0283, Rv0284, Rv0285, Rv0286, Rv0287, Rv0288, Rv0289, Rv0290, Rv0291 and Rv0292. In a most preferred embodiment, the deletion of the complete esx-3 region renders the resultant recombinant M. tuberculosis less virulent than the wildtype. In an embodiment, the Rv0285 gene is a PE5 gene. In an embodiment, the Rv0286 gene is a PPE4 gene. In an embodiment, the Rv0287 gene is a EsxG gene. In an embodiment, the Rv0288 gene is a EsxH gene. The Rv numbered genes can be identified as set forth in databases of the Mycobacterium tuberculosis H37Rv genome (for example, see tuberculist.epfl.ch, genome.tbdb.org, or see the annotations of the genes as set forth in NCBI Reference Sequence: NC_(—)000962.3).

Further M. tuberculosis genome sequences are known in the art and can be found, for example, at Genbank, (www.ncbi.nlm.nih.gov/genbank/). Sequences corresponding to the ESAT-6 gene cluster region 3 (esx-3 region), e.g. identified by Rv0282 through Rv0292 in H37Rv, are readily identifiable by those of ordinary skill in the art, for example by using widely-available sequence alignment software tools. In an embodiment, the invention encompasses recombinant M. tuberculosis comprising a deletion in genomic sequences corresponding to the ESAT-6 gene cluster region 3 (esx-3 region).

The non-naturally occurring mutant Mycobacterium tuberculosis bacterium may be created so as to comprise further advantageous mutations known in the art that confer reduced virulence, which render the bacterium auxotrophic for an amino acid or for a vitamin (e.g. delta panCD, delta RD1 and delta leuCd mutants), which promote Th1 cytokine profile, and/or which increase the ability of the bacterium to induce apoptosis of a mammalian macrophage. Non-limiting examples of further mutations which can be incorporated into the mutant bacteria of the invention include NuoG mutations, NlaA mutations (see, e.g., US 2010/0297185, Jacobs et al., published Nov. 25, 2010, hereby incorporated by reference), SecA2 mutations (e.g. see U.S. Pat. No. 8,101,191, issued Jan. 24, 2012, Jacobs et al., hereby incorporated by reference) and region of difference 1 (RD1) mutations (e.g. see U.S. Pat. No. 7,722,861, Jan. 24, 2003, Jacobs et al.). In an embodiment, the aforementioned mutants are deletion mutants.

In a preferred embodiment of the mutant bacteria, the non-naturally occurring mutant Mycobacterium tuberculosis bacterium is viable, is live and/or is capable or propagating. In an embodiment, the mutant is capable of propagating. In an embodiment, the mutant is capable of propagating in a medium comprising a non-mycobactin dependent source of iron. In an embodiment, the mutant is recoverable in the presence of a non-mycobactin dependent source of iron in its environment for propagation. Examples of non-mycobactin dependent sources of iron are provided herein. In an embodiment, the mutant is capable of propagating in a solid medium comprising a mycobactin wherein the mycobactin is at a concentration of at least 200 ng/ml. In an embodiment, the mutant is not capable of propagating in a solid medium comprising a mycobactin wherein the mycobactin is at a concentration of 2 ng/ml or 20 ng/ml. In an embodiment, the solid media is 7H10 media supplemented with mycobactin J (Allied Monitor).

The mutant M. tuberculosis of any of Claims 1-5, wherein the mutant requires for propagation presence either of (i) a non-mycobactin dependent source of iron in its environment, or (ii) a mycobactin source of iron at least 200 ng/ml in its environment.

In an embodiment, the mutant the genome of the mutant is complemented with a nucleic acid sequence identical to an ESAT-6 gene cluster region 3 (esx-3 region) of a genome of mycobacterium which is not a M. tuberculosis. In an embodiment, the mycobacterium which is not a M. tuberculosis is a M. smegmatis.

A method is provided of producing the mutant Mycobacterium tuberculosis bacteria of the invention is also provided. In an embodiment, the mutant Mycobacterium tuberculosis bacterium comprising a deletion in the esx-3 region of the genome is made by a method comprising deleting a nucleic acid sequence in the esx-3 region of a genome of a M. tuberculosis bacterium by homologous recombination with a non-replicating plasmid which plasmid comprises (i) a nucleic acid sequence identical to a portion of the genome immediately upstream of the deleted nucleic acid sequence and (ii) a nucleic acid sequence identical to a portion of the genome immediately downstream of the deleted nucleic acid sequence, but (iii) no portion identical to the deleted region, in the presence of a non-mycobactin dependent source of iron, or in the presence of a mycobactin dependent source of iron wherein the mycobactin is present at a concentration of 200 ng/ml or greater. In an embodiment, the method is in the presence of a non-mycobactin dependent source of iron.

A method is also provided of producing a mutant Mycobacterium tuberculosis bacterium, wherein the mutant M. tuberculosis comprises a deletion in the esx-3 region of a genome of the M. tuberculosis bacterium, the method comprising deleting a nucleic acid sequence in the esx-3 region of a genome of a M. tuberculosis bacterium. In an embodiment, the method comprises effecting deleting the nucleic acid sequence in the esx-3 region of a genome by homologous recombination. In an embodiment, the homologous recombination is performed with a non-replicating plasmid which plasmid comprises (i) a nucleic acid sequence homologous to a portion of the genome immediately upstream of the deleted nucleic acid sequence and (ii) a nucleic acid sequence homologous to a portion of the genome immediately downstream of the deleted nucleic acid sequence, but (iii) no portion identical to the deleted region. In an embodiment, the homologous recombination is performed with a non-replicating plasmid which plasmid comprises (i) a nucleic acid sequence identical in sequence to a portion of the genome immediately upstream of the deleted nucleic acid sequence and (ii) a nucleic acid sequence identical in sequence to a portion of the genome immediately downstream of the deleted nucleic acid sequence. In an embodiment, the method is performed in the presence of a non-mycobactin dependent source of iron. In an embodiment, the method is performed in the presence of a mycobactin dependent source of iron wherein the mycobactin is present at a concentration of 200 ng/ml or greater. In an embodiment of the methods, the method further comprises recovering the mutant M. tuberculosis. In an embodiment of the methods, the method further comprises maintaining the mutant M. tuberculosis in the presence of a non-mycobactin dependent source of iron. In an embodiment of the methods, the method further comprises maintaining the mutant M. tuberculosis in the presence of mycobactin at a concentration of 200 ng/ml or greater.

In an embodiment of the methods, homologous recombination with the non-replicating plasmid is effected by introducing the plasmid into the M. tuberculosis bacterium by way of a transducing phage. Reference (1), the contents of which are hereby incorporated by reference in their entirety, provides an overview of molecular biology techniques that can be employed in the methods herein or producing the mutant M. tuberculosis. In an embodiment, the transducing phage described herein is a mycobacteriophage. As used herein, a “mycobacteriophage” is a phage capable of infecting one or more mycobacteria. Background art for the concept of producing recombinant or mutant mycobacteriophages which may be used in the methods of the invention of producing the mutant M. tuberculosis are discussed in U.S. Pat. No. 6,300,061, and shuttle phasmids are discussed in U.S. Pat. No. 5,750,384, both of which patents are incorporated by reference in their entirety.

In a preferred embodiment of the methods, the transducing phage comprises a phAE159 vector comprising the plasmid sequence. The phage phAE159 is a useful vector for the use in the methods of the invention. The phAE159 has a high cloning capacity and is derived from the temperature sensitive ph101 vector which in turn is derived from TM4. As such, phAE159 is useful as a vector backbone of the invention. In a preferred embodiment, the vector backbone is a phAE159 vector. In an embodiment, the phAE159 vector backbone comprises the sequence set forth in SEQ ID NO:5. In another embodiment, the vector backbone is a ph101 vector. Phages derived from TM4 which are useful as embodiments of the vector backbone in the present invention include, in non-limiting examples, those set forth in Genbank Accession No. JF937104: JF704106: JF704105, HM152764: and HM152767.

In a preferred embodiment of the methods, the non-replicating plasmid further comprises a nucleic acid sequence which provides the recombinant Mycobacterium tuberculosis bacterium with resistance to an anti-bacterial antibiotic. A preferred antibiotic resistance gene is a hygromycin resistance gene. A further example is an ampicillin resistance gene.

In a preferred embodiment of the methods, the genome of the mutant is complemented with a nucleic acid sequence identical to an ESAT-6 gene cluster region 3 (esx-3 region) of a genome of mycobacterium which is not a M. tuberculosis. In an embodiment, the mycobacterium which is not a M. tuberculosis is a M. smegmatis.

In a preferred embodiment of the methods, the non-replicating plasmid further comprises a nucleic acid sequence which encodes a detectable marker. In embodiments, the detectable marker is a β-galactosidase encoded by a LacZ, a maltose binding protein, a chloramphenicol acetyltransferase, or a fluorescent protein. In an embodiment, the fluorescent protein is a green or yellow fluorescent protein. In a further embodiment, the fluorescent protein is green fluorescent protein derived from A. victoria. Other useful elements, commonly known in the art, may also be included in the genome of the recombinant mycobacteria of the invention. The recombinant mycobacteriophages and vectors of the invention can optionally comprise a mycobacteriophage integration sequence. A sacB gene may be included so as to facilitate unmarking (the sacB gene is from Bacillus subtilis, and is inducible by sucrose and lethal when expressed in Gram-negative bacteria).

In an embodiment of the mutants, compositions, and/or methods, the non-mycobactin dependent source of iron comprises hemin, transferrin, lactoferrin, or ferritin. In a preferred embodiment, the non-mycobactin dependent source of iron comprises hemin. In an embodiment of the mutants, compositions, and/or methods, the non-mycobactin dependent source of iron comprises hemoglobin, whole blood (e.g. blood agar plates), or lysed erythrocytes (e.g. chocolate agar plates). In an embodiment, the methods are performed in the presence of mycobactin or carboxymycobactin. In an embodiment, the mycobactin or carboxymycobactin is present in the media at a concentration of 200 ng/ml or more. In an embodiment, the medium is a solid medium.

In an embodiment of the methods, the concentration of hemin in the culture media in which the mutant Mycobacterium tuberculosis bacteria are maintained is sufficient to maintain viability of the mutant Mycobacterium tuberculosis bacteria. In an embodiment, the concentration of hemin the culture media in which the mutant Mycobacterium tuberculosis bacteria are maintained is in excess of 10 μM. In an embodiment, the concentration of hemin the culture media in which the mutant Mycobacterium tuberculosis bacteria are maintained is in excess of 25 μM, 25 μM-50 μM, 50 μM-75 μM, 75 μM-100 μM, 100 μM-150 μM, 150 μM-200 μM, or in excess of 200 μM. In a preferred embodiment, the concentration of hemin the culture media in which the mutant Mycobacterium tuberculosis bacteria is between 90 μM and 100 μM, is about 100 μM, or is 100 μM.

In an embodiment of the instant method, the sequence homologous to, or identical to, a portion of the genome immediately upstream of the deleted nucleic acid sequence is 825-950 basepairs in length. In a preferred embodiment of the instant method, the sequence is 900 basepairs in length. In an embodiment, the sequence the sequence homologous to, or identical to, a portion of the genome immediately downstream of the deleted nucleic acid sequence is 825-950 basepairs in length. In a preferred embodiment, the sequence is 867 basepairs in length. In an embodiment, the sequence in the plasmid identical to a portion of the genome immediately upstream of the deleted nucleic acid sequence is contiguous with the sequence identical to a portion of the genome immediately downstream of the deleted nucleic acid sequence.

In embodiments, the mutant M. tuberculosis created by the methods described herein is any of the mutant M. tuberculosis described herein, including, for example, those created so as to comprise further advantageous mutations.

Also provided is a composition comprising a mutant Mycobacterium tuberculosis bacterium, wherein the mutant M. tuberculosis comprises a deletion in the esx-3 region of a genome of the M. tuberculosis bacterium and a carrier. In an embodiment, the carrier is a pharmaceutically acceptable carrier. In an embodiment, the composition further comprises an immunological adjuvant. In an embodiment, the mutant Mycobacterium tuberculosis bacterium is live. In an embodiment, the mutant Mycobacterium tuberculosis bacterium is capable or propagating. In an embodiment, the carrier comprises a culture media. In an embodiment, the carrier comprises a non-mycobactin dependent source of iron as described herein. In an embodiment, the non-mycobactin dependent source of iron comprises hemin. In an embodiment, the composition is a vaccine composition. In an embodiment, the carrier comprises mycobactin or carboxymycobactin of at least 200 ng/ml.

Compositions are also provided by the invention, comprising any of the mutant Mycobacterium tuberculosis bacteria described herein, wherein the mutant M. tuberculosis comprises a deletion in the esx-3 region of a genome of the M. tuberculosis bacterium and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are known in the art and can be chosen based on intended use. In an embodiment, the genome of the mutant is complemented with a nucleic acid sequence identical to an ESAT-6 gene cluster region 3 (esx-3 region) of a genome of mycobacterium which is not a M. tuberculosis. In an embodiment, the mycobacterium which is not a M. tuberculosis is a M. smegmatis.

In an embodiment, wherein the composition is intended for administration to a subject, the composition further comprises an immunological adjuvant. Immunological adjuvants encompassed within the compositions and methods of the invention are widely known in the art and include alum, other aluminum salts (e.g. aluminum phosphate and aluminum hydroxide) and squalene. Other immunological adjuvants encompassed within the compositions and methods of the invention include the compounds QS21 and MF59. In an embodiment, the composition is vaccine. In an embodiment, the composition is a live vaccine. In an embodiment, the vaccine comprises a live mutant Mycobacterium tuberculosis bacteria described herein. In an embodiment, the vaccine comprises a pharmaceutically acceptable carrier. In an embodiment, the vaccine further comprises an immunological adjuvant.

Any of the compositions of the invention, or any of the mutant M. tuberculosis bacteria of the invention, can be used to evoke an immune response in a subject. In an embodiment, administration of a composition of the invention, or the naked mutant M. tuberculosis bacteria of the invention, is used to elicit an immune response in the subject. In an embodiment, the eliciting an immune response in a subject is effected by a method comprising administering to the subject a composition comprising a non-naturally occurring mutant Mycobacterium tuberculosis bacterium, wherein the mutant M. tuberculosis comprises a deletion in the esx-3 region of a genome thereof, in an amount effective to elicit an immune response. In a preferred embodiment, the mutant M. tuberculosis comprises a deletion of the esx-3 region (Rv0282-Rv0292) of the genome. In a preferred embodiment, the composition comprises an immunological adjuvant.

The invention also provides a non-naturally occurring mutant Mycobacterium tuberculosis bacterium, wherein the mutant M. tuberculosis comprises a deletion of (i) EsxG, (ii) EsxH, (iii) Esxg and EsxH, (iv) PE5-PPE4, or (v) the four genes PE5 to esxH, of the genome of the M. tuberculosis bacterium. The invention also provides a composition comprising such. In an embodiment the M. tuberculosis bacterium is an H37Rv strain. In an embodiment, the mutant further comprises deletion of a gene of the genome involved in amino acid synthesis or involved in vitamin synthesis. In an embodiment, the non-naturally occurring mutant comprises a deletion of a RD1 encoding gene, a LeuCD encoding gene, and/or a panCD encoding gene. A composition comprising the non-naturally occurring mutant of any of Claims 47-49 and a carrier. In an embodiment, the composition is a vaccine composition. In an embodiment, the composition comprises an immunological adjuvant.

The invention also provides a method of eliciting an immune response in a subject comprising administering to the subject a composition comprising a non-naturally occurring mutant Mycobacterium tuberculosis bacterium, or the composition comprising such, wherein the non-naturally occurring mutant M. tuberculosis comprises a deletion of (i) esxG, (ii) esxH, (iii) esxg and esxH, (iv) PE5-PPE4, or (v) the four genes PE5 to esxH, of a genome of the M. tuberculosis bacterium, in an amount effective to elicit an immune response. In an embodiment the M. tuberculosis bacterium is an H37Rv strain. In an embodiment, the mutant further comprises deletion of a gene of the genome involved in amino acid synthesis or involved in vitamin synthesis. In an embodiment, the non-naturally occurring mutant comprises a deletion of a RD1 encoding gene, a LeuCD encoding gene, and/or a panCD encoding gene.

In a preferred embodiment of the mutants, compositions and methods of the inventions described herein, the M. tuberculosis bacterium is an H37Rv strain. For H37Rv genome, see NCBI Reference Sequence: NC_(—)000962.2, see GenBank: AL123456.2.

The methods disclosed herein involving subjects can be used with any species capable of being infected by M. tuberculosis. In a preferred embodiment, the subject is a mammalian subject. Most preferably, the mammal is a human.

All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

Experimental Results

A strategy was investigated for to recovering viable M. tuberculosis esx-3 deletion mutants. It was hypothesized that the presence of hemin in the culture media might provide such a route. The entire esx-3 region was deleted from the M. tuberculosis H37Rv strain via homologous recombination using specialized transducing phage. The deletion phasmid for the Δesx-3 mutant was constructed by PCR amplification of the 5′-flanking region of Rv0282 using M. tuberculosis H37Rv genomic DNA as template with the following primer pairs: esx-3LL 5′ TTTTTTTTCCATAAATTGGTGGCGGCGGGGCTGGACTC 3′ (SEQ ID NO:1) and esx-3LR 5′ TTTTTTTCCATTTCTTGGCCACGCCTCCGCTGTCTCCTTC 3′ (SEQ ID NO:2). The PCR product was 900 bp. For the 3′-flanking region of Rv0292, the following primer pairs were used: esx-3RL 5′ TTTTTTTTCCATAGATTGGGGCTGCACTGGCCTACTCCTAC 3′ (SEQ ID NO:3) and esx-3RR 5′ TTTTTTTTCCATCTTTTGG-GCGCCAGCGGTGGAGTGCATTG 3′ (SEQ ID NO:4). This PCR product was 867 bp. Following cloning into plasmid p0004S (esx-3.p0004S) containing the hygromycin resistance cassette and the sacB gene to facilitate unmarking (2), the resulting plasmid was then packaged into the temperature-sensitive phage phAE159, as described earlier (1), to yield the knockout phage for esx-3. Specialized transduction was performed, as described previously (1) and the transduction mix was spread on 7H10 plates, selecting with 50 μg/ml hygromycin. Additionally, plates were supplemented with iron and zinc at various concentrations, and with hemin at 10 μM or 100 μM. After four weeks of incubation at 37° C., colonies were present only on the 7H10 plates supplemented with hemin at 100 μM (FIG. 1).

Confirmation of the successful knockout of the M. tuberculosis esx-3 region: To confirm that the colonies obtained were esx-3 deletion mutants, genomic DNA was prepared from H37Rv Δesx-3 transductants (#1-#3) and H37Rv wild-type. The DNA was digested with BamHI and, after separation by agarose gel electrophoresis, Southern blots were performed, using as probes sequences immediately flanking the deleted esx-3 region: a 900-bp left flank probe amplified with primers esx-3LL and esx-3LR (panel A) or a 867-bp right flank probe amplified with primers esx-3RL and esx-3RR (panel B). Analysis of the results demonstrated that both the left and right flank probes hybridized with DNA fragments of sizes expected for the knockout strain and clearly distinct from those expected of the parental strain (FIGS. 2A and 1B). The size differences are the result of elimination of BamHI sites from the chromosome by the extensive deletion, while the inserted sacB-hygromycin resistance cassette contains no BamHI sites (shown schematically in FIGS. 2C and 2D).

That the recovered colonies were in fact esx-3 deletion mutants was further confirmed by PCR analysis. Genomic DNA was prepared from either the H37Rv Δesx-3 transductants, or from the parental H37Rv wt strain, and was used as template in PCR reactions to amplify products using three different sets of primers (FIG. 3). Primer set P1+P2+P3 includes a common left hand primer, P1, to the left of the cloned flank sequence, binding in both wt and mutant, while complementary primers P2 (specific for wild-type) and P3 (specific for mutant) yield distinct product sizes for mutant and wild-type. Similarly, primer set P4+P5+P6 includes a common right hand primer, P4, to the right of the cloned flank sequence, and complementary primers P5 (specific for wild-type) and P6 (specific for mutant). Finally, primer set P7+P8 includes primers entirely within the deleted esx-3 region, and so amplifies a product in wild-type only. Schematics of H37Rv wt (FIG. 3A) and H37Rv Δesx-3 (FIG. 3B) chromosomal loci indicate the locations of the three primer sets, and the expected product sizes for wild-type and mutant. As shown in FIG. 3C, PCR products confirmed the recovery of the Δesx3 mutants as these samples yielded products corresponding to the expected size for the deletion mutant and a size clearly distinct from that obtained using wild-type genomic DNA. As expected, a water control yielded no product. These data demonstrate that Δesx-3 strains were successfully recovered with the use of hemin supplementation. Subsequent studies demonstrated that mycobactin J at 200 ng/ml or 2000 ng/ml also supports Δesx-3 strain growth on agar. This demonstrates that Δesx-3 strains are viable, in contrast to the current understanding in the art, and that the esx-3 locus appears to be “conditionally essential”, and may be deleted when an alternative source of iron (either mycobactin or a non-mycobactin dependent source) is provided.

Growth of the Δesx-3 strain is enhanced in vitro by supplementation of growth medium with hemin: It was hypothesized that an M. tuberculosis Δesx-3 strain would exhibit impaired growth in the absence of hemin. As noted, following transduction H37Rv Δesx-3 colonies were obtained on 7H10 agar supplemented with hemin at 100 μM (FIG. 1) at 4 weeks of incubation, but not on 7H10 lacking hemin (not shown). Once the mutants were obtained and confirmed, studies were undertaken to further explore the effect of hemin on growth. The H37Rv Δesx-3 strain, cultured in the presence of hemin 100 μM, was washed, diluted and plated onto 7H10 agar with and without hemin, in comparison with the H37Rv wt parental strain. Hemin was demonstrated to enhance growth of the Δesx-3 strains relative to the parental strain on solid medium 7H10 (FIG. 4). Similarly, growth in liquid 7H9 medium was also enhanced by supplementation with hemin at the highest concentration studied, 100 μM (FIG. 5). The fact that growth is seen in liquid media even in the absence of hemin (as well as on solid media at lower dilutions—not shown) may be due to carryover of hemin from the original culture medium, or perhaps to intracellular storage of iron. A similar phenomenon has been observed by others for M. bovis BCG mutants in the mycobactin synthesis pathway, which are able to grow for some generations in the absence of exogenous mycobactin—the higher the concentration of mycobactin in the original culture medium, the more growth (doublings) is observed in the absence of mycobactin (8). Similarly, growth in liquid 7H9 medium was also enhanced by supplementation with hemin at the highest concentration studied, 100 μM (FIG. 5). Again, growth is observed in the absence of exogenous hemin, equivalent to ˜6 doublings (from OD₆₀₀ ˜0.025 to OD₆₀₀ ˜1.6), perhaps due to the intracellular storage issues mentioned above. The 7H9 medium used in these studies is also complex, containing BSA supplementation which may potentially contain some heme.

Using specialized transduction, we have generated strains deleted for esxG (Rv0287) and esxH (Rv0288). Like the Δesx-3 strain, these mutants grow when provided supplemental mycobactin J (FIG. 8). These data demonstrate that loci within the Esx3 region that do not encompass the entire Esx3 region are also conditionally essential. Utilizing specialized transduction, we have also generated strains harboring combined deletions of esxG-esxH (Rv0287-Rv0288) and PE5-PPE4 (Rv0285-Rv0286), and PE5 through esxH (Rv0285-Rv0288) and were able to recover the deletion mutants on medium supplemented with mycobactin J at 2000 ng/ml.

The Δesx3 strain is highly attenuated following iv inoculation into SCID mice, RAG(−/−) mice and MyD88 (−/−) mice: The esx-3 region of the soil-dwelling organism Mycobacterium smegmatis has recently been deleted from the chromosome, and the resulting strain complemented by chromosomal integration of the orthologous M. tuberculosis esx-3 region (6). This yielded the IKEPLUS strain, named for its phenotypes with respect to “Immune Killing Evasion” (6). M. smegmatis IKEPLUS was found to be significantly attenuated (similar to the parental M. smegmatis Δesx-3 IKE strain from which it derived), and additionally was found to induce a highly potent T helper type I (T_(H)1) cytokine response in infected mice, with enhanced production of IL-12 and IFN-γ, and very low IL-6 compared with wild-type M. smegmatis. As predicted from this cytokine milieu, which should provide efficient priming of T_(H)1 cell responses, IKEPLUS was found to be a highly effective vaccine against challenge with virulent M. tuberculosis. Mice vaccinated with IKEPLUS exhibited prolonged survival following M. tuberculosis challenge, as compared with BCG-vaccinated mice, and IKEPLUS vaccination also induced significant declines in bacterial burdens in the tissues of mice surviving to later time points after challenge, declines which in some cases exceeded by 3 logs those seen in BCG-immunized mice, and in one experiment actually resulted in sterilizing immunity. The M. tuberculosis genes present in IKEPLUS were essential to produce this highly effective immune response, as protection was much more modest with the parental IKE strain, with survival and bacterial burden similar to naïve mice (6).

Given the above summarized findings regarding attenuation of the IKE and IKEPLUS strains, the virulence of the H37Rv Δesx-3 strain was assessed. SCID mice (7 to 8 per group) were infected intravenously with a high dose, 10⁷ (by OD₆₀₀ estimate), of wasH37hed bacilli, and survival was monitored over time (FIG. 6). Very significant attenuation of the Δesx-3 strain was apparent, as all of the H37Rv wild-type-infected mice succumbed to the infection within 16 days, while all Δesx-3-infected mice remain alive and without signs of illness at >50 days after infection.

The M. tuberculosis Δesx-3 strains disclosed herein can be used in live attenuated tuberculosis vaccines and also as the backbone for live attenuated tuberculosis vaccines which include additional attenuating and/or immunomodulatory mutations. The H37Rv Δesx3 strain was used to subcutaneously vaccinate immunocompetent C57BL/6 mice at a dose of 10⁶/mouse. Controls included unvaccinated (Naïve) animals and animals vaccinated with BCG, and another candidate vaccine strain H37Rv ΔleuDpanCDsecA2. The animals were challenged by low dose aerosol infection with M. tuberculosis Erdman strain at two months post vaccinationBacterial burdens in lung and spleen were determined at 1, 3 and 5 months post-challenge (Table 1). In addition, animals were challenged 6 months post-vaccination and lung and spleen titers were determined one month post-challenge (Table 1). At all time points examined, the Δesx-3 vaccine reduced lung titers by a statistically-significant amount. In addition, Δesx-3 vaccine reduced spleen titers to a statistically-significant degree in the spleen at one month post-challenge in animals challenged either one or six months after vaccination. These data support the use of Δesx-3 as a vaccine or as a backbone for a vaccine.

FIG. 8 shows Mid- to late-log phase cultures of the indicated bacterial strains were washed in PBS+0.05% tyloxapol, resuspended in volumes to give equivalent OD₆₀₀ values, then serially diluted in PBS-tyoxapol to give approximately 300 colonies per 100 μl, and 100 μl aliquots were plated onto each plate. The strains included H37Rv Δesx-3, H37Rv Δesx-3 attB::pYUB1335 (complemented with a cosmid containing the esx-3 region of M. tuberculosis H37Rv), H37Rv Δesx-3 attB::pYUB2076 (complemented with a cosmid containing the paralogous esx-3 region of M. smegmatis), H37RvΔesxG (Rv0287), H37RvΔesxH (Rv0288), and H37RvΔmbtB (Rv2383c). It can be observed that growth of the Δesx-3, ΔesxG and ΔesxH mutants is not supported in 7H10 media, but significant growth is observed on 7H10 media supplemented with mycobactin J (Allied Monitor) at 200 ng/ml. In contrast, the ΔmbtB mutant, predicted from the literature to be deficient in mycobactin synthesis, is able to grow with mycobactin J supplement in amounts as low as 2 ng/ml. The ΔesxG mutant is unique among these mutants in the ability to grow on 7H10 supplemented with additional iron and zinc: in this case ferric ammonium citrate (FAC) at 250 μg/ml and zinc sulfate (ZnSO₄) at 10 μg/ml.

FIG. 9 shows a PCR Screen for H37Rv Δesx-3 complemented with a cosmid encoding the M. smegmatis esx-3 region (pYUB2076).

TABLE 1 Bacterial burden (log₁₀ cfu) in lung and spleen after subcutaneous vaccination with the indicated strains followed by aerosol challenge with M. tuberculosis Erdman strain. Experimental Group Lung Spleen 2 months post-vaccination, 1 month post-challenge Naive 5.88 ± 0.05  5.00 ± 0.06 BCG 5.02 ± 0.06*  4.14 ± 0.04* ΔleuDpanCDsecA2 5.48 ± 0.10*  4.61 ± 0.14* Δesx-3 4.86 ± 0.11*  4.03 ± 0.13* 2 months post vaccination, 3 month post challenge Naive 5.31 ± 0.20  4.71 ± 0.25 BCG 4.83 ± 0.26* 4.01 ± 0.61 ΔleuDpanCDsecA2 5.22 ± 0.12  4.67 ± 0.17 Δesx-3 4.82 ± 0.25* 4.73 ± 0.44 2 months post vaccination, 5 months post challenge Naive 5.44 ± 0.16  4.41 ± 0.08 BCG 5.15 ± 0.16  4.34 ± 0.04 ΔleuDpanCDsecA2 5.36 ± 0.14  4.36 ± 0.03 Δesx-3 5.04 ± 0.07* 4.27 ± 0.14 6 months post vaccination, 1 month post challenge Naive 5.88 ± 0.03  4.62 ± 0.06 BCG 4.91 ± 0.03*  4.15 ± 0.09* ΔleuDpanCDsecA2 5.04 ± 0.03*  4.16 ± 0.04* Δesx-3 4.72 ± 0.14*  4.08 ± 0.03* *Statistically different than naïve controls (p < 0.05)

REFERENCES

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1. A non-naturally occurring mutant Mycobacterium tuberculosis bacterium, wherein the mutant M. tuberculosis comprises a deletion in the ESAT-6 gene cluster region 3 (esx-3 region) of a genome of the M. tuberculosis bacterium.
 2. The mutant M. tuberculosis of claim 1, wherein the mutant M. tuberculosis comprises a deletion of the esx-3 region (Rv0282 through Rv0292) of the genome.
 3. The mutant M. tuberculosis of claim 1, wherein the M. tuberculosis bacterium is an H37Rv strain.
 4. The mutant M. tuberculosis of claim 1, wherein the mutant is capable of propagating.
 5. The mutant M. tuberculosis of claim 1 wherein the mutant is capable of propagating in a medium comprising a non-mycobactin dependent source of iron.
 6. The mutant M. tuberculosis of claim 1, wherein the mutant requires presence of a non-mycobactin dependent source of iron in its environment for propagation.
 7. A method of producing the non-naturally occurring mutant Mycobacterium tuberculosis bacterium of claim 1, the method comprising deleting a nucleic acid sequence in the esx-3 region of a genome of a M. tuberculosis bacterium.
 8. The method of claim 7, comprising effecting deletion of the nucleic acid sequence in the esx-3 region of the genome by homologous recombination.
 9. The method of claim 8, wherein homologous recombination is performed with a non-replicating plasmid which plasmid comprises (i) a nucleic acid sequence homologous to a portion of the genome immediately upstream of the nucleic acid sequence being deleted and (ii) a nucleic acid sequence homologous to a portion of the genome immediately downstream of the nucleic acid sequence being deleted, but (iii) no portion identical to the deleted region.
 10. The method of claim 8, wherein homologous recombination is performed with a non-replicating plasmid which plasmid comprises (i) a nucleic acid sequence identical in sequence to a portion of the genome immediately upstream of the deleted nucleic acid sequence and (ii) a nucleic acid sequence identical in sequence to a portion of the genome immediately downstream of the deleted nucleic acid sequence.
 11. The method of claim 7, wherein method is performed in the presence of a non-mycobactin dependent source of iron. 12-15. (canceled)
 16. The method of claim 9, wherein homologous recombination with the non-replicating plasmid is effected by introducing the plasmid into the M. tuberculosis bacterium by way of a transducing phage.
 17. The method of claim 16, wherein the transducing phage comprises a phAE159 vector comprising the plasmid sequence. 18-22. (canceled)
 23. A composition comprising a mutant Mycobacterium tuberculosis bacterium, wherein the mutant M. tuberculosis comprises a deletion in the esx-3 region of a genome of the M. tuberculosis bacterium and a carrier.
 24. The composition of claim 23, wherein the carrier is a pharmaceutically acceptable carrier.
 25. The composition of claim 23, further comprising an immunological adjuvant.
 26. The composition of claim 23, wherein the mutant Mycobacterium tuberculosis bacterium is live. 27-30. (canceled)
 31. The composition of claim 23 which is a vaccine composition.
 32. A method of eliciting an immune response in a subject comprising administering to the subject a composition comprising a non-naturally occurring mutant Mycobacterium tuberculosis bacterium, or the composition of claim 23, wherein the mutant M. tuberculosis comprises a deletion in the esx-3 region of a genome thereof, in an amount effective to elicit an immune response.
 33. The method of claim 32, wherein the mutant M. tuberculosis comprises a deletion of the esx-3 region (Rv0282-Rv0292) of the genome. 34-53. (canceled) 